The overall objective of this proposal is to develop a method to study both the cellular localization and quantity of amino acids in the same brain. A procedure will be developed for immunocytochemical staining of sections of tissue fixed on the slide rather than by perfusion-fixation of the animal. This method will be applied in this study to localization of immunoreactivity to certain amino acids (glutamate, glycine, GABA), but it could be applied to localization of other substances as well, such as enzymes, peptides, catecholamines, serotonin, etc. The purpose in developing this procedure is to allow sections adjacent to those stained immunocytochemically to be dissected into microscopic-sized samples and quantitatively assayed for the same substances as those stained. In this way, the strengths of both approaches can be applied to particular problems, with one method providing quantitation, with a lower degree of localization, and the other providing localization, with a lower degree of quantitation. Using the technique of "quantitative histochemistry" (Lowry, J. Histochem. Cytochem. 1:420-428, 1953), samples as small as 20mum on a linear side may be dissected from freeze-dried sections, weighed, and quantitatively assayed for concentrations of free amino acids by high performance liquid chromatography (HPLC). Precise locations of microdissected samples will be mapped so that exact comparisons with adjacent sections be used to calibrate image analysis measurements of stained sections. Background staining, non-specific crossreactive staining (false positives), and poor immunoreactivity (false negatives) can be better identified as such by correlation of stained sections with adjacent ones dissected and assayed for amino acid concentrations. The two methods, quantitative histochemistry and immunocytochemistry, are complementary in that the weaknesses of one are the strengths of the other. With one method providing quantitation and the other localization, it would be good to use both approaches, particularly in studies dealing with dynamic changes, such as occurs during development, aging, response to lesions, plasticity, and other situations. The tissues chosen for analysis in this study would be hindbrain areas related to vestibular function in guinea pig, including the vestibular nuclear complex and layers of the cerebellum. The data obtained would serve as a pilot study to be extended to investigating neurochemical compensatory changes following unilateral labyrinthectomy.